Changes in Arabidopsis thaliana Gene Expression in Response to

Aug 20, 2013 - Expression Microarray, Fox Chase Cancer Center, Philadelphia, Pennsylvania ... ABSTRACT: The release of silver nanoparticles (AgNPs) in...
0 downloads 0 Views 1MB Size
Subscriber access provided by University of Rochester | River Campus & Miner Libraries

Article

Changes in Arabidopsis thaliana gene expression in response to silver nanoparticles and silver ions Rashid Kaveh, Yue-Sheng Li, Sibia Ranjbar, Rouzbeh Tehrani, Christopher L. Brueck, and Benoit Van Aken Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es402209w • Publication Date (Web): 20 Aug 2013 Downloaded from http://pubs.acs.org on August 25, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Environmental Science & Technology

1

Changes in Arabidopsis thaliana gene expression in response to silver nanoparticles and

2

silver ions

3 4

Rashid Kaveh†, Yue-Sheng Li‡, Sibia Ranjbar†, Rouzbeh Tehrani†, Christopher L. Brueck†,

5

Benoit Van Aken†,*

6 7



Pennsylvania 19122, United States

8 9

Department of Civil and Environmental Engineering, Temple University, Philadelphia,



Expression Microarray, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, United States

10 11 12

* Corresponding author phone: 215-204-7087; fax: 215-204-4696; e-mail: [email protected]

13 14 15

Abstract: The release of silver nanoparticles (AgNPs) in the environment has raised concerns

16

about their effects on living organisms, including plants. In this study, changes in gene

17

expression in Arabidopsis thaliana exposed to polyvinylpyrrolidone-coated AgNPs and silver

18

ions (Ag+) were analyzed using Affymetrix expression microarrays. Exposure to 5 mg AgNPs L-

19

1

20

reference to non-exposed plants. Exposure to 5 mg Ag+ L-1 for 10 days resulted in up-regulation

21

of 84 genes and down-regulation of 53 genes by reference to non-exposed plants. Many genes

22

differentially expressed by AgNPs and Ag+ were found to be involved in plant response to

23

various stresses: up-regulated genes were primarily associated with response to metals and

(20 nm) for 10 days resulted in up-regulation of 286 genes and down-regulation of 81 genes by

1

ACS Paragon Plus Environment

Environmental Science & Technology

24

oxidative stress (e.g., vacuolar cation/proton exchanger, superoxide dismutase, cytochrome P-

25

450-dependent oxidase, and peroxidase), while down-regulated genes were more associated with

26

response to pathogens and hormonal stimuli (e.g., auxin-regulated gene involved in organ size

27

(ARGOS), ethylene signaling pathway, and systemic acquired resistance (SAR) against fungi

28

and bacteria). A significant overlap was observed between genes differentially expressed in

29

response to AgNPs and Ag+ (13% and 21% of total up- and down-regulated genes, respectively),

30

suggesting that AgNP-induced stress originates partly from silver toxicity and partly from

31

nanoparticle-specific effects. Three highly up-regulated genes in the presence of AgNPs, but not

32

Ag+, belong to the thalianol biosynthetic pathway, which is thought to be involved in plant

33

defense system. Results from this study provide insights into the molecular mechanisms of plant

34

response to AgNPs and Ag+.

35 36

Keywords: Silver nanoparticles, silver, Arabidopsis thaliana, gene expression, microarray

37 38

TOC/Abstract Art

39

40 41 2

ACS Paragon Plus Environment

Page 2 of 31

Page 3 of 31

Environmental Science & Technology

42

Introduction

43

Engineered nanoparticles (ENPs) are utilized in an increasing number of products, including

44

textiles, electronics, pharmaceuticals, cosmetics, and water treatment reagents.1 ENPs have

45

recently raised environmental concerns because they are likely to be released into the

46

environment as a consequence of their widespread utilization and because they have been shown

47

to exert particle-size specific toxic effects on most living organisms.2,3 Effective regulation of the

48

release of ENPs in the environment has been slow to emerge, because of the relatively recent

49

recognition of their environmental effects, the broad range of their application (e.g., industry,

50

cosmetics, medicine), and the lack of characterization of their toxic effects.4,5 In the U.S., the

51

utilization and potential release of ENPs are regulated through the Environmental Protection

52

Agency (EPA) Toxic Substances Control Act (TSCA). Although EPA is currently developing a

53

Significant New Use Rule (SNUR) to ensure that nanoscale materials receive appropriate

54

regulatory review, it appears that there is currently no regulation specific to the utilization,

55

release, and maximum acceptable levels of ENPs in the environment. There is therefore a critical

56

need to collect more experimental data about the ecotoxicity of different kinds of ENPs to

57

support further regulatory efforts from federal agencies. Silver nanoparticles (AgNPs) constitute

58

the most widely used ENPs. Primarily because of their antimicrobial properties, AgNPs have

59

been used in a wide variety of products, including textiles, bandages, deodorants, baby products,

60

toothpaste, air filters, and house appliances.6,7 The ecotoxicology of AgNPs is complex because

61

it may be related simultaneously to silver-specific and nanoparticle-specific biological effects.8,9

62

Both AgNPs and dissolved silver have been shown to be toxic for bacteria, algae, aquatic

63

organisms, plants, and humans.10,11,12 As plants constitute the basis of the terrestrial food chain,

64

their exposure to AgNPs has potential implications for the agriculture and human health.10

3

ACS Paragon Plus Environment

Environmental Science & Technology

65

AgNPs released from consumer products are likely to enter wastewater. In wastewater treatment

66

plants, AgNPs partition between biosolids and treated water, which can be applied on

67

agricultural fields through fertilization or irrigation processes.13,14 Potential uptake of AgNPs by

68

agricultural plants has therefore raised concerns about contamination of the food chain, including

69

humans. As observed with other kinds of ENPs, prior studies focusing on the exposure of plants

70

to AgNPs have reported both positive effects (hormeosis) and negative effects (toxicity).6,7,8,9,15

71 72

Most toxicological studies on the effects of AgNPs have been conducted through acute toxicity

73

testing (short-time exposure to high dose) although environmental effects are more adequately

74

assessed by chronic toxicity testing (long-time exposure to low dose). Although chronic toxicity

75

is typically difficult to observe in the laboratory, molecular studies (e.g., proteomic or

76

transcriptomic methods) may provide useful information about the potential long-term effects of

77

exposure to environmental contaminants.16 However, the molecular bases of the toxicity and/or

78

growth promoting effects of AgNPs in higher plants have received little attention. Recently,

79

changes in gene expression in plants exposed to various environmental contaminants have been

80

studied using the microarray technology, providing better understanding of the molecular

81

mechanisms of plant response.17-20 Following a similar strategy, the objective of this study is to

82

provide new insights on the transcriptional response of the model plant, Arabidopsis thaliana,

83

exposed to AgNPs and Ag+ through the use of whole-genome cDNA microarrays.

84 85

Experimental

86

Chemicals. Silver nanoparticles (silver nanopowder, 99.99%, 20 nm, CAS 7440-22-4) were

87

obtained from U.S. Research Nanomaterials (Houston, TX). Silver nitrate (99.9%) was obtained

4

ACS Paragon Plus Environment

Page 4 of 31

Page 5 of 31

Environmental Science & Technology

88

from Sigma-Aldrich (St-Louis, MO). Polyvinylpyrrolidone (molecular weight 40,000 Dalton,

89

PVP40) was obtained from Sigma-Aldrich. Phytoagar was obtained from Plant Media (Dublin,

90

OH). Murashige and Skoog (MS) salt base was obtained from Carolina (Burlington, NC). Other

91

chemicals were of analytical grade, solvents were of HPLC grade, and they were obtained from

92

Acros Chemicals (Geel, Belgium), Fischer Scientific (Pittsburgh, PA), or Sigma-Aldrich.

93 94

Nanoparticle characterization: For AgNP characterization, fresh particle suspensions were

95

prepared in 0.5-strength Murashige and Skoog (MS) medium (at concentration of 5 to 20 mg L-1

96

with an equal concentration of PVP40) and dispersed by sonication for 30 min in a water bath

97

(150 W). Particle suspensions were analyzed immediately after preparation and after 24 h of

98

agitation at 55 °C, 150 rpm. Particle suspensions were characterized by visible spectrometry,

99

dynamic light scattering (DLS), and transmission electron microscopy (TEM). Visible spectra

100

(300 to 800 nm) of particle suspensions were recorded using an Agilent 8453 spectrophotometer

101

(Agilent, Santa Clara, CA). The particle size distribution and zeta potential of the suspensions

102

were determined by DLS using a Zetasizer Nano (Malvern, Worcester, MA) with the following

103

parameters: wavelength 632.8 nm, angle 173 °, temperature 25 °C. The particle size and

104

morphology were characterized by TEM using a JEM-1400 (JEOL, Peabody, MA) with a HT

105

voltage of 120 kV and a beam current of 66 mA. Samples were prepared by applying and air

106

drying 4 µL of particle suspension onto a 400-mesh copper grid covered with ultrathin carbon

107

film on Holey carbon support film (Ted Pella, Redding, CA).

108 109

Plant species and culture conditions. Arabidopsis thaliana, ecotype Columbia (Col-0/Redei-

110

L211497), was obtained from the Arabidopsis Biological Resource Center (Ohio State

5

ACS Paragon Plus Environment

Environmental Science & Technology

111

University, Columbus, OH). Seeds were kept on a wet filter paper at 4 ºC in the dark for 24 h.

112

Seeds were then surface-sterilized by immersion successively in DI water for 1 h, in 95% ethanol

113

for 5 min, and 0.6% sodium hypochloride for 5 min, and were rinsed 3 times in sterile DI water.

114

Seeds were germinated under sterile conditions in 10 × 10-cm Magenta boxes (5 seeds per box)

115

closed with vented lids and filled with 100 mL of semi-solid nutrient medium. The medium

116

consisted of 0.5-strength MS nutrient solution supplemented with 0.3% sucrose and 0.7%

117

phytoagar (pH 7.2), and it was sterilized by autoclaving (121 °C, 15 min). The boxes were

118

incubated at 25 ºC under white (cool) fluorescent light (0.38 ± 0.02 W ft-2) with a 16-h light/8-h

119

dark photoperiod.

120 121

Growth inhibition experiments. The inhibitory effect of AgNPs and Ag+ was tested by cultivating

122

Arabidopsis plants in the presence of increasing concentrations of the toxicants (1.0, 2.5, 5.0, 10,

123

and 20 mg L-1) added to the nutrient medium. After sterilization, the medium (prepared as

124

described above) was cooled to 55 ºC and supplemented with the AgNPs or Ag+ (formulated as

125

aqueous stock solutions). AgNP stock solution was prepared by mixing 1.0 g AgNPs L-1 (0.1%

126

w/v) and 1.0 g PVP40 L-1 (0.1% w/v) with DI water and dispersing AgNPs by sonication for 30

127

min in a water bath (150 W). Ag+ stock solution was prepared by dissolving 1.575 g AgNO3 L-1

128

(0.1% w/v Ag) and 1.0 g PVP40 L-1 (0.1% w/v) in DI water. Two sets of non-exposed control

129

plants were grown in nutrient medium (MS) only and in nutrient medium supplemented with 20

130

mg PVP40 L-1 (i.e., equivalent to the highest concentration of PVP40 applied in the experiments

131

with AgNPs). After 10 days of growth, plants were removed from the medium, washed with

132

water to remove the excess of medium, dried by blotting, and weighted. Twenty plants were used

133

for each treatment. The significance of differences between treatments was evaluated using one-

6

ACS Paragon Plus Environment

Page 6 of 31

Page 7 of 31

Environmental Science & Technology

134

way ANOVA (Prim 6.0, GraphPad, La Jolla, CA) followed by Tukey's multiple comparison tests

135

at 95% confidence level (p < 0.05).

136 137

Analysis of silver in plant tissues: For the determination of silver content, plants were grown and

138

exposed to AgNPs and Ag+ as described above. At harvesting time, plants were washed with

139

deionized water to remove silver that was neither adsorbed nor integrated in plant tissues.1 Plants

140

were then separated into roots and leaves, dried at 70 °C for 24 h, and digested in 4:1

141

concentrated HNO3:30% H2O2 at 70 °C for 8 h. After dilution with deionized water, the solution

142

was filtrated through 0.2 µm and analyzed using an Agilent 7500i Benchtop ICP-MS (Santa

143

Clara, CA). Fifteen plants were used for each treatment. Leaf or root tissues from 5 plants were

144

pooled together to obtain three biological replicates that were analyzed separately. Statistical

145

analyses were performed using one-way ANOVA followed by Tukey's multiple comparison

146

tests.

147 148

Gene expression analysis using microarrays. Plants were grown as described above in the

149

presence of 5 mg AgNPs or Ag+ L-1 (and, in each case, 5 mg PVP40 L-1). Control plants were

150

grown in the presence of 5 mg PVP40 L-1 only. After 10 days of growth, plants were removed

151

from the medium, washed, immediately soaked in 4 mL of RNA Later™ (Ambion, Foster City,

152

CA), and incubated at 4 ºC for 24 h following the manufacturer's guidelines. The plants were

153

then dried by blotting and stored at -80 ºC until RNA extraction. RNA was extracted from whole

154

plant tissues using TRIzol® Plus RNA Purification kit with on-column PureLink® DNAase

155

treatment following the manufacturer's guidelines. An additional step of tissue homogenization

156

was performed after addition of the TRIzol® reagent using bead beating (1-mm glass beads,

7

ACS Paragon Plus Environment

Environmental Science & Technology

157

4,200 rpm, 40 sec). Purified RNA was kept at -80 °C. RNA was quantified by the OD260 using a

158

NanoDrop™ ND-2000 spectrophotometer (Vernon Hills, IL). The quality of RNA was assessed

159

by the ratios OD260/OD280 and OD260/OD230 and using an Agilent 2100 Bioanalyzer (Santa Clara,

160

CA). RNA samples used for microarray analysis had OD260/OD280 ratios of 2.12 – 2.17,

161

OD260/OD230 ratios of 2.16 – 2.36, and RNA integrity numbers (RIN) of 8.3 – 9.1. RNA samples

162

were labeled and hybridized to the Affymetrix Arabidopsis Gene 1.0 ST Arrays according to the

163

manufacturer's instructions (Affymetrix, Santa Clara, CA). For each treatment, three biological

164

replicate samples were used for microarray experiments. Scanned microarray images were

165

analyzed using the Affymetrix Gene Expression Console with RMA (Robust Multi-array

166

Average) normalization algorithm. Further statistical analyses were performed using BRB-

167

ArrayTools developed by Dr. Richard Simon and BRB-ArrayTools Development Team.21 Gene

168

classification into ontology categories (GO) was performed using BLAST2GO® version 2.6.4

169

(Biobam Bioinformatics, Valencia, Spain).

170 171

Reverse-transcription real-time PCR. Quantitative analysis of gene expression was performed

172

for selected genes using reverse-transcription real-time PCR (RT-qPCR). Four genes

173

significantly down-regulated (fold change < 0.25), 4 genes significantly up-regulated (fold

174

change > 4.0), and 4 genes with moderate expression differences (fold change between 0.4 and

175

2.0) upon exposure to both AgNPs and Ag+ were selected (Supplemental Information, Table S1).

176

The internal standard was the housekeeping gene, mitogen-activated protein kinase 6 (MPK6)

177

(AT2G43790).20 RT-qPCRs were conducted for three biological replicates per treatment using

178

the same RNA as used for the microarray experiments. RNA was reverse-transcribed into cDNA

179

using SuperScript® III First-Strand Synthesis system and oligo-dT primers (Invitrogen, Foster

8

ACS Paragon Plus Environment

Page 8 of 31

Page 9 of 31

Environmental Science & Technology

180

City, CA). Negative controls were generated by running the reactions without reverse-

181

transcriptase. Gene sequences were obtained from the National Center for Biotechnology

182

Information (NCBI) and used to design gene-specific real-time primers using PrimerQuest (IDT,

183

Coralville, IA). When possible (for 10 of the 12 selected genes), primers were designed with one

184

of the primer sequence spanning an exon-intron boundary (primer sequences are provided as

185

Supplemental Information, Table S1). Real-time PCR quantification of cDNA was performed on

186

a StepOnePlus™ Real-Time PCR System using SYBR® Green PCR Master Mix (Applied

187

Biosystems, Foster City, CA). The amplification efficiency for each primer set was determined

188

by using log10-dilutions of cDNA according to standard protocols. CT (cycle threshold) data were

189

computed by the StepOnePlus™ Software (version 2.1; Applied Biosystems). The mean relative

190

levels of amplification of the target genes and standard deviations were calculated based on CT

191

values and amplification efficiencies using REST 2009 (version 2.0.13; Qiagen, Foster City,

192

CA).22

193 194

MIAME compliance. This article is written in compliance with the Minimum Information About

195

a Microarray Experiment (MIAME) guidelines (http://www.mged.org/miame). Microarray data

196

have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) with

197

the accession number E-MEXP-3950.

198 199

Results and Discussion

200

Nanoparticle characterization: Particle suspensions were characterized in fresh preparation (MS

201

medium) and after 24 h of agitation at 55 °C, 150 rpm to detect potential aggregation and/or

202

change in properties. The spectrum of the fresh AgNP suspension (5 mg AgNPs L-1, 5 mg

9

ACS Paragon Plus Environment

Environmental Science & Technology

203

PVP40 L-1 in 0.5-strength MS medium) showed a single peak at 405 nm, which is consistent

204

with uniform-sized nanoparticles with a diameter of approx. 20 nm. No significant change of the

205

spectrum was observed after agitation of the suspension for 24 h at 55 °C, 150 rpm (spectra are

206

presented in Supplemental Information, Figure S1). DLS analysis of the fresh AgNP suspension

207

(20 mg L-1) showed a narrow size distribution with a hydrodynamic diameter of 27.1 ± 0.3 nm,

208

which shifted to 29.6 ± 0.1 nm after 24 h of agitation at 55 °C. The zeta potential of the fresh

209

suspension was -38.1 ± 0.3 V, which shifted to -30.9 ± 0.6 V after 24 h of agitation at 55 °C (size

210

distribution diagrams are presented in Supplemental Information, Figure S2). TEM pictures of

211

AgNP suspensions showed mostly spherical particles of uniform size (Figure 1). No observable

212

change was recorded after agitation at 55 °C for 24 h.

213 214

Effect of AgNPs and Ag+ on A. thaliana growth. In order to determine the potential toxic effect

215

of AgNPs and Ag+ on A. thaliana, plantlets were grown in MS medium containing increasing

216

concentration of both factors, ranging from 0.0 to 20 mg L-1. Figure 2 presents the average fresh

217

biomass of plants exposed for 10 days to each treatment. No significant biomass difference was

218

observed between the sets of non-exposed control plants (i.e., growing in MS medium only and

219

in MS medium containing 20 mg PVP40 L-1). Exposure of plants to 1.0 and 2.5 mg AgNPs L-1

220

for 10 days resulted in significant increase of the biomass, although exposure to higher

221

concentrations (from 5.0 to 20 mg L-1) resulted in reduction of the biomass. On the other hand,

222

although no significant effect was recorded in the presence of 1.0 and 2.5 mg Ag+ L-1, the

223

biomass after 10 days was significantly reduced upon exposure to 5.0 mg L-1 and above. At low

224

levels (1.0 and 2.5 mg L-1), exposure to AgNPs resulted in a significantly higher plant biomass

10

ACS Paragon Plus Environment

Page 10 of 31

Page 11 of 31

Environmental Science & Technology

225

than exposure to Ag+. No significant difference was recorded between the two treatments at

226

higher levels (5.0, 10, and 20 mg L-1).

227 228

Previous studies focusing on the impact of AgNPs on plants have reported both positive and

229

negative effects. Investigating the impact of AgNPs of different sizes (from 2 to 20 nm) in barley

230

(Hordeum vulgare), flax (Linum usitatissimum), and ryegrass (Lolium perenne), El-Temsah and

231

Joner6 reported a significant reduction of the germination and growth rates, which were

232

dependent on the plant species and concentration of AgNPs: e.g., 20-nm particles resulted in a

233

noticeable reduction of the shoot length at 10 mg L-1 in flax and ryegrass, and at 20 mg L-1 in all

234

three species. Another study reported a limited effect of AgNPs (approx. 30 nm) on cucumber

235

(Cucumis sativus) and lettuce (Lactuca sativa) plants.15 Germination rates were reduced by 24%

236

and 5% and root elongation rates were reduced by 15% and 2% after exposure to 100 mg AgNPs

237

L-1 in cucumber and lettuce, respectively. Kumari et al.7 studied the cytotoxicity and

238

genotoxicity of 100-nm AgNPs on onion root tip cells (Allium cepa): based on microscopic

239

observations, cells exposed to increasing concentration in AgNPs (from 25 to 100 mg L-1)

240

showed a decrease of the mitotic index (MI) from 60% to 28% and the occurrence chromosomal

241

aberrations. Although previous studies reported mostly toxic effects of AgNPs toward plants, a

242

recent report from Wang et al.9 showed that exposure of Arabidopsis and poplar (Populus

243

deltoides × nigra DN34) plants to AgNPs produced beneficial effects at low concentration: e.g.,

244

exposure to 25-nm AgNPs resulted in increased biomass and evapotranspiration in poplars at the

245

concentration of 1.0 mg L-1, although exposure to 10 and 100 mg L-1 resulted in decrease of

246

these parameters.

247

11

ACS Paragon Plus Environment

Environmental Science & Technology

248

Analysis of silver content in plant tissues: After 10 days of exposure to AgNPs and Ag+, plant

249

tissues were dried, acid-digested, and the silver content was determined by ICP-MS. At the same

250

level of exposure, the Ag concentration was significantly higher in plants dosed with Ag+ than in

251

plants dosed with AgNPs. The Ag concentration was also significantly higher in exposed root

252

tissues than in exposed leave tissues. A positive relationship was observed between the exposure

253

dose and Ag content in root tissues, although this relationship was less apparent in exposed leaf

254

tissues. Overall, these results are consistent with previous studies described in the literature. As

255

an example, Wang et al.9 recently reported a higher Ag content in A. thaliana leaves exposed to

256

Ag+ (approx. 8 µg g-1) than exposed to AgNPs (approx. 2 µg g-1). As observed in our study, the

257

Ag concentration detected in roots was higher than in aerial parts of the plants: 68% of total Ag+

258

and 24% of total AgNPs were found in root tissues, while only 2% of total Ag+ and 1% of total

259

AgNPs were found in aerial parts.

260 261

Gene expression microarrays. The transcriptional response of plants exposed to AgNPs and Ag+

262

was investigated using Affymetrix whole-transcript expression microarrays. AgNP and Ag+

263

concentration of 5 mg L-1, which resulted in moderate reduction of the plant biomass, was

264

chosen for microarray experiments. After filtering out the genes with low-quality signals and

265

conducting univariate t-tests (p < 0.001; BRB-ArrayTools), 446 and 405 genes showed

266

consistent expression levels after exposure to AgNPs and Ag+, respectively. Of these, 375 and

267

141 genes were expressed at significantly different levels (change fold < 0.5 or > 2.0) by

268

exposure to AgNPs and Ag+, respectively: 286 genes (78%) were up-regulated and 81 genes

269

(22%) were down-regulated at significant levels by exposure to AgNPs; 84 genes (60%) were

270

up-regulated and 53 genes (40%) were down-regulated at significant levels by exposure to Ag+.

12

ACS Paragon Plus Environment

Page 12 of 31

Page 13 of 31

Environmental Science & Technology

271

A significant overlap of differentially-expressed genes was observed upon exposure to AgNPs

272

and Ag+: 15 genes were up-regulated and 29 genes were down-regulated in response to both

273

AgNPs and Ag+ (representing 13% and 21% of the total up- and down-regulated genes,

274

respectively). This observation suggests that some of the AgNP effects on gene expression

275

originate from Ag+ released by AgNPs, which was previously observed or proposed by other

276

authors.9,25 As in several other studies9,23,24, we chose to expose the plants to the same

277

concentrations of AgNPs and Ag+ (expressed as mg Ag L-1). This approach was motivated by the

278

lack of information about the nature of the toxic effects and cellular targets of both Ag forms.

279

Soluble Ag is known to be cytotoxic and AgNP toxicity likely originates partly from the release

280

of soluble Ag. Besides, nanoparticles are known to exert specific 'particulate' effects that may not

281

be related to Ag+ toxicity. Dimkpa et al.25 reported that exposure of sand-grown wheat plants

282

(Triticum aestivum) to both 10-nm AgNPs and soluble Ag (Ag+) (2.5 mg kg-1) resulted in

283

comparable biomass reduction. Interestingly, exposure to Ag+ equivalent to soluble Ag released

284

from AgNPs (63 µg kg-1) did not result in observable effect. Moreover, using TEM, the authors

285

observed accumulation of AgNPs inside plant tissues, suggesting that AgNPs exert toxic effects

286

that are, at least in part, unrelated to the release of soluble Ag. In order to determine the

287

respective effect of AgNPs and Ag+, Stampoulis et al.8 exposed zucchini plants (Cucurbita pepo)

288

to bulk (powder) Ag, AgNPs (100 nm), and Ag+ (both supernatant of AgNP suspension and

289

AgNO3). The authors reported that exposure to bulk Ag at 1,000 mg L-1 did not significantly

290

affect the plant biomass, while exposure to 1,000 mg L-1 AgNPs reduced the biomass by approx.

291

75%. On the other hand, AgNP supernatant and Ag+ (AgNO3) at concentration as low as 1.0 mg

292

L-1 reduced the biomass by approx. 25%, suggesting that about half the observed phytotoxicity

293

originated from the elemental nanoparticles themselves. Few studies have been conducted on the

13

ACS Paragon Plus Environment

Environmental Science & Technology

294

transcriptional response of organisms exposed to AgNPs. Analyzing gene expression in human

295

cells (HeLa) exposed to AgNPs and Ag+, Xu et al.12 reported a higher number of genes up-

296

regulated (62%) than down-regulated (38%) with Ag nanoparticle exposure, which is consistent

297

with our results. The authors also observed that a large number of genes were differentially

298

expressed in response to both AgNPs and Ag+ (85% of up-regulated genes and 68% of down-

299

regulated genes). The complete list of Arabidopsis genes up- and down-regulated by exposure to

300

AgNPs and Ag+ is provided as Supplemental Information (Tables S2 and S3).

301 302

Reverse-transcription real-time PCR. In order to validate the microarray results, quantitative

303

analysis of gene expression was performed on selected genes using RT-qPCR. Figure 4 shows

304

the plots of the expression levels of the selected genes as recorded using microarrays against

305

their expression levels recorded using RT-qPCR. Correlations were generally satisfactory with

306

Pearson's correlation coefficient of 0.96 and 0.97 for exposure to AgNPs and Ag+, respectively.

307

The RT-qPCR amplification levels were corrected for the amplification efficiencies of different

308

primer sets (ranging from 97.9% to 109.9%, R2 = 0.99 to 1.0) using REST 2009.

309 310

Functional categories of differentially expressed genes. Differentially expressed genes were

311

classified in gene ontology (GO) categories using the software BLAST2GO®. Distribution into

312

major process and functional categories (GO level 2) showed little difference between genes up-

313

and down-regulated by exposure to AgNPs and Ag+: most represented process categories for

314

both up- and down-regulated genes were metabolic process, cellular process, response to

315

stimulus, and biological regulation (Figure 5); most represented functional categories for both

316

up- and down-regulated genes were catalytic activity, binding, nucleic acid binding transcription

14

ACS Paragon Plus Environment

Page 14 of 31

Page 15 of 31

Environmental Science & Technology

317

factor activity, and transporter activity (Figure 6). Two remarkable exceptions are the signaling

318

process category involving only down-regulated genes (25% and 30% of total down-regulated

319

genes by exposure to AgNPs and Ag+, respectively) and the electron carrier functional category

320

involving (almost) only up-regulated genes (11% and 16% of total up-regulated genes by

321

exposure to AgNPs and Ag+, respectively). Genes in the signaling category are mostly involved

322

in hormone signaling pathways and cellular response to hormone stimuli, which may be related

323

to the reduction of plant growth in response to the toxicity of AgNPs and Ag+.26 On the other

324

hand, heavy metals are known to interact with electron carriers. Up-regulation of genes in this

325

category may reflect the response of the plant to the decreased electron transport efficiency in the

326

presence of AgNPs and Ag+.27

327 328

A significant proportion of genes differentially expressed by exposure to AgNPs and/or Ag+ is

329

involved in response to stimuli (GO level 2) (49% of total up-regulated genes and 68% of total

330

down-regulated genes). Among them, most up-regulated genes (63% and 78% of genes in this

331

category up-regulated by AgNPs and Ag+, respectively) are involved in response to abiotic

332

stimuli (GO level 5), including metal ions, salts, light, starvation, oxidative stress, osmotic stress,

333

and radiation. On the other hand, most down-regulated genes (74% and 62% of genes in this

334

category down-regulated by AgNPs and Ag+, respectively) are involved in response to pathogens

335

and hormonal stimuli (GO level 5), including abscisic acid, auxine, cytokinin, ethylene,

336

gibberellin, jasmonic acid, and steroid hormones (Figure 6). Up-regulation of genes related to

337

abiotic stimuli likely reflects the response of the plant to AgNP/Ag+-induced stress. On the other

338

hand, down-regulation of genes related to hormones and biological stimuli can be seen as a plant

339

strategy to prioritize the response to AgNP and Ag+-induced stress.26 Alternatively, hormones are

15

ACS Paragon Plus Environment

Environmental Science & Technology

340

involved primarily in the regulation of plant development and down-regulation of hormone-

341

responsive genes may simply reflect attempts of the plant to limit the growth under toxic

342

conditions. In addition, beside their role in plant development, hormones are known to be

343

involved in response to biological and abiotic stresses. For instance, the down-regulation of

344

abscisic acid and auxin signaling pathways has been shown to play a role in various plant

345

defense responses.26

346 347

Differential expression of remarkable genes. Remarkable genes discussed in this section were

348

differentially expressed in response to both AgNPs and Ag+ (reflecting the effects of Ag+) or in

349

response to AgNPs only (reflecting nanoparticle-specific effects). Genes highly up-regulated

350

(fold change > 4.0) in response to both AgNPs and Ag+ are involved in response to metal and

351

oxidative stresses. These genes encode the following proteins: a vacuolar cation/proton

352

exchanger involved in root development under metal stress (AT5G01490), a miraculin-like

353

protein (MLP) (AT2G01520), two copper/zinc superoxide dismutases (AT2G28190 and

354

AT1G08830), two cytochrome P-450-dependent monooxygenases (AT5G42590 and

355

AT3G28740), and a peroxidase (AT3G21770). MLPs have been suggested to be involved in

356

response to wounding and pathogen infection in other plant species.28 Superoxide dismutases and

357

peroxidases are involved in protection against reactive oxygen species (ROS), which are

358

frequently associated with metal and ENP toxicity.1 Although they are involved in many

359

physiological processes, cytochrome P-450 genes have been reported to be induced by metal

360

stress in Arabidopsis.29 Genes most down-regulated in response to both AgNPs and Ag+ (fold

361

change ≤ 0.25) include a gene encoding an ARGOS (auxin-regulated gene involved in organ

362

size) protein (AT3G59900), three genes involved in the ethylene signaling pathway

16

ACS Paragon Plus Environment

Page 16 of 31

Page 17 of 31

Environmental Science & Technology

363

(AT3G16770, AT5G25350, and AT2G40940), and two genes involved in systemic acquired

364

resistance (SAR) against fungi (AT4G12470) and bacteria (AT5G46330). The ARGOS gene (the

365

most down-regulated gene by both AgNP and Ag+, 0.08- and 0.09-fold change, respectively)

366

regulates the size of lateral organs and its down-regulation likely reflects attempts of the plant to

367

limit its expansion under stressed conditions.26 As suggested above, down-regulation of genes

368

involved in ethylene signaling pathway and SAR can be understood as a prioritization of plant

369

defense mechanisms under AgNP and Ag+-induced stresses.26 Genes differentially expressed in

370

response to both AgNPs and Ag+ are likely to be implicated in response to Ag+, either directly

371

added or released from AgNPs.

372 373

On the other hand, a number of genes were differentially expressed in response to AgNPs only,

374

which reflects their involvement in nanoparticle-specific responses. The most remarkable genes

375

up-regulated (fold change > 4.0) specifically by AgNPs include two genes involved in salt stress

376

(AT3G28220 and AT1G52000), a gene encoding a myrosinase-binding protein involved in

377

defense against insects and pathogens (AT1G52040), three genes involved in the thalianol

378

biosynthetic pathway (AT5G48010, AT5G48000, and AT5G47990), and a gene encoding a MLP

379

involved in response to wounding (AT2G01520). The most up-regulated gene in our study (28.6-

380

fold change) encodes a TRAF (tumor necrosis factor receptor-associated factor)-like protein

381

involved in salt stress response. Although the relationship between salt and AgNP-induced stress

382

is not readily apparent, similar functional genes were found to be induced in Arabidopsis

383

exposed to other kinds of ENPs.20 The induction of genes responsive to pathogens and wounding

384

may be related to mechanical damages caused by AgNPs to plant tissues.3 The three genes of the

385

thalianol pathway belong to a cluster of four genes, which constitutes a rare case of gene

17

ACS Paragon Plus Environment

Environmental Science & Technology

386

clustering in higher plants.30 Although gene clusters are common in bacteria (i.e., operons), they

387

are less frequent in eukaryotes and were thought until recently to be restricted to paralogs

388

originating from repeated tandem genes. Only a few clusters of non-homologous, functionally-

389

related genes have been detected in fungi and plants. In plants, these clusters are all involved in

390

biosynthesis of stress-induced secondary metabolites that are (or are believed to be) required for

391

survival under specific conditions, such as the exploitation of new environments.31

392 393

Interestingly, the most down-regulated gene by exposure to both AgNP and Ag+ in our study

394

(ARGOS) was also reported down-regulated in Arabidopsis plants exposed to zinc oxide and

395

fullerene nanoparticles.20 Other remarkable genes up-regulated by both AgNPs (our study) and

396

zinc oxide nanoparticles20 are genes encoding a superoxide dismutase (AT1G08830) and two

397

peroxidases, which are involved in response to oxidative stress (AT3G21770, AT2G18150), and

398

a phytosulfokine-beta growth factor involved in response to wounding (AT3G49780).

399 400

This article presents the first whole-genome expression microarray experiment focusing on A.

401

thaliana plants exposed to AgNPs and Ag+. Results from this study are believed to provide new

402

insights into the molecular mechanisms of plant response to AgNPs and Ag+.

403 404

Acknowledgments

405

This research was supported by a U.S. Department of Agriculture (USDA) - National Institute of

406

Food and Agriculture (NIFA) grant (Award number: 2012-67009-19982).

407 408

Supplemental Information

18

ACS Paragon Plus Environment

Page 18 of 31

Page 19 of 31

Environmental Science & Technology

409

The genes selected for the RT-qPCR validation of microarray results and the corresponding

410

primer sequences are listed in Table S1. The complete list of Arabidopsis genes significantly up-

411

regulated (> 2.0) and down-regulated (< 0.5) by exposure to AgNPs and Ag+ is provided in

412

Tables S2 and S3. Visible absorption spectra and DLS size-distribution diagrams of AgNP

413

suspensions are presented in Figure S1 and Figure S2, respectively. This information is available

414

free of charge via the Internet at http://pubs.acs.org/.

415 416

References

417

1. Navarro, E.; Baun, A.; Behra, R.; Hartmann, N.B.; Filser, J.; Miao, A.; Quigg, A.; Santschi,

418

P.H.; Sigg, L. Environmental behavior and ecotoxicity of engineered nanoparticles to algae,

419

plants, and fungi. Ecotoxicology 2008, 17, 372-386.

420

2. Klaine, S.J.; Alvarez, P.J.J.; Batley, G.E.; Fernandes, T.F.; Handy, R.D.; Lyon, D.Y.;

421

Mahendra, S.; McLaughlin, M.J.; Lead, J.R. Nanomaterials in the environment: Behavior,

422

fate, bioavailability, and effects. Environ. Toxicol. Chem. 2008, 27, 1825-1851.

423

3. Handy, R.D.; Cornelis, G.; Fernandes, T.; Tsyusko, O.; Decho, A.; Sabo-Attwood, T.;

424

Metcalfe, C.; Steevens, J.A.; Klaine, S.J.; Koelmans, A.A.; Horne, N. Ecotoxicity test

425

methods for engineered nanomaterials: Practical experiences and recommendations from the

426

bench. Environ. Toxicol. Chem. 2012, 31, 15-31.

427 428 429

4. Seaton, A.: Tran, L.; Aitken, R.; Donaldson, K. Nanoparticles, human health hazard and regulation. J. R. Soc. Interface 2009, doi:10.1098/rsif.2009.0252.focus. 5. Auffan, M.; Rose, J.; Bottero, J.-Y.; Lowry, G.V.; Jolivet, J.-P., Wiesner, M.R. Towards a

430

definition of inorganic nanoparticles from an environmental, health and safety perspective.

431

Nat. Nanotechnol. 2009, 4, 634-641.

19

ACS Paragon Plus Environment

Environmental Science & Technology

432

6. El-Temsah, Y.S.; Joner, E.J. Impact of Fe and Ag nanoparticles on seed germination and

433

differences in bioavailability during exposure in aqueous suspension and soil. Environ.

434

Toxicol. 2012, 27, 42-49.

435 436 437 438

7. Kumari, M.; Mukherjee, A.; Chandrasekaran, N. Genotoxicity of silver nanoparticles in Allium cepa. Sci. Total Environ. 2009, 407, 5243-5246. 8. Stampoulis, D.; Sinha, S.K.; White, J.C. Assay-dependent phytotoxicity of nanoparticles to plants. Environ. Sci. Technol. 2009, 43, 9473-9479.

439

9. Wang, J.; Koo, Y.; Alexander, A.; Yang, Y.; Westerhof, S.; Zhang, Q.; Schnoor, J.L.; Colvin,

440

V.L.; Braam, J.; Alvarez, P.J.J. Phytostimulation of poplars and Arabidopsis exposed to silver

441

nanoparticles and Ag+ at sublethal concentrations. Environ. Sci. Technol. 2013,

442

doi.org/10.1021/es4004334.

443

10. Ma, X.; Geiser-Lee, J.; Deng, Y.; Kolmakov, A. Interactions between engineered

444

nanoparticles (ENPs) and plants: Phytotoxicity, uptake and accumulation. Sci. Total Environ.

445

2010, 408, 3053-3061.

446

11. Fabrega, J.; Luoma, S.N.; Tyler, C.R.; Galloway, T.S.; Lead, J.R. Silver nanoparticles:

447

Behaviour and effects in the aquatic environment. Environ. Int. 2011, 37, 517-531.

448 449

12. Xu, L.; Takemura, T.; Xu, M.; Hanagata, N. Toxicity of silver nanoparticles as assessed by global gene expression analysis. Materials Express 2011, 1, 74-79.

450

13. Blaser S.A; , Scheringer, M., MacLeod, M.; Hungerbühler K. Estimation of cumulative

451

aquatic exposure and risk due to silver: Contribution of nano-functionalized plastics and

452

textiles. Sci. Tot. Environ. 2008, 390, 396 – 409.

20

ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31

Environmental Science & Technology

453

14. Kaegi, R.; Voegelin, A.; Sinnet, B.; Zuleeg, S.; Hagendorfer, H.; Burkhardt, M.; Siegrist, H.

454

Behavior of metallic silver nanoparticles in a pilot wastewater treatment plant. Environ. Sci.

455

Technol. 2011, 45, 3902-3908.

456 457 458

15. Barrena, R.; Casals, E.; Colon, J.; Font, X.; Sanchez, A.; Puntes, V. Evaluation of the ecotoxicity of model nanoparticles. Chemosphere 2009, 75, 850-857. 16. Ankley ,G.T.; Daston ,G.P.; Degitz , S.J.; Denslow , N.D.; Hoke , R.A.; Kennedy , S.W.;

459

Miracle, A.L.; Perkins , E.J.; Snape , J.; Tillitt , D.E.; Tyler, C.R.; Versteeg, D.

460

Toxicogenomics in regulatory ecotoxicology. Environ. Sci. Technol. 2006, 13, 4055-4065.

461

17. Mentewab, A.; Cardoza, V.; Stewart, C.N. Genomic analysis of the response of Arabidopsis

462

thaliana to trinitrotoluene as revealed by cDNA microarrays. Plant Sci. 2005, 168, 1409-

463

1424.

464

18. Gandia-Herrero, F.; Lorenz, A.; Larson, T.; Graham, I.A.; Bowles, D.J.; Rylott, E.L.; Bruce,

465

N.C. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis: Discovery of

466

bifunctional O- and C-glucosyltransferases. Plant Journal 2008, 56, 963-974.

467

19. Jin, X.; Shuai, J.; Peng, R.; Zhu, B.; Fu, X.; Tian, Y.; Zhao, W.; Han, H.; Chen, C.; Xu, J.;

468

Yao, Q.; Qu, S.; Xiong, A. Identification of candidate genes involved in responses of

469

Arabidopsis to polychlorinated biphenyls based on microarray analysis. Plant Growth

470

Regulation 2011, 65, 127-135.

471

20. Landa, P.; Vankova, R.; Andrlova, J.; Hodek, J.; Marsik, P.; Storchova, H.; White, J.C.;

472

Vanek, T. Nanoparticle-specific changes in Arabidopsis thaliana gene expression after

473

exposure to ZnO, TiO2, and fullerene soot. J. Hazard. Mater. 2012, 241, 55-62.

474

21. Simon, R.; Lam, A.; Li, M.-C.; Ngan, M.; Menenzes, S.; Zhao, Y. Analysis of gene

475

expression data using BRB-Array Tools. Cancer Inform. 2007, 3, 11-17.

21

ACS Paragon Plus Environment

Environmental Science & Technology

476

22. Pfaffl, M.W.; Horgan, G.W.; Dempfle, L. Relative expression software tool (REST) for

477

group-wise comparison and statistical analysis of relative expression results in real-time PCR.

478

Nucleic Acids Res. 2002, 30, e36.

479

23. Jiang, H.-S.; Li, M.; Chang F.-Y.; Li, W.; Yin, L.-Y. Physiological analysis of silver

480

nanoparticles and AgNO3 toxicity to Spirodela polyrhiza. Environ. Toxicol. Chem. 2012, 31,

481

1880-1886.

482

24. Yasur, J.; Rani P.U. Environmental effects of nanosilver: Impact on castor seed germination,

483

seedling growth, and plant physiology. Environ. Sci. Pollut. Res. 2013, doi 10.1007/s11356-

484

013-1798-3.

485

25. Dimkpa, C.O.; McLean, J.E.; Martineau, N.; Britt, D.W.; Haverkamp, R.; Anderson, A.J.

486

Silver nanoparticles disrupt wheat (Triticum aestivum L.) growth in a sand matrix. Environ.

487

Sci. Technol. 2013, 47, 1082-1090.

488 489 490 491 492

26. Bari, R.; Jones, J.D.G. Role of plant hormones in plant defence responses. Plant Mol. Biol. 2009, 69:473-488. 27. Prasad, M.N.; Strzalka, K. Physiology and Biochemistry of Metal Toxicity and Tolerance in Plants. Springer: New York, 2002; p 462. 28. Tsukuda, S.; Gomi, K.; Yamamoto, H.; Akimitsu, K. Characterization of cDNAs encoding

493

two distinct miraculin-like proteins and stress-related modulation of the corresponding

494

mRNAs in Citrus jambhiri Lush. Plant Mol. Biol. 2006, 60,125-136.

495

29. Narusaka, Y.; Narusaka, M.; Seki, M.; Umezawa, T.; Ishida, J.; Nakajima, M.; Enju, A.;

496

Shinozaki, K. Crosstalk in the responses to abiotic and biotic stresses in Arabidopsis:

497

Analysis of gene expression in cytochrome P450 gene superfamily by cDNA microarray.

498

Plant Mol. Biol. 2004, 55, 327-342.

22

ACS Paragon Plus Environment

Page 22 of 31

Page 23 of 31

499 500 501 502

Environmental Science & Technology

30. Field, B.; Osbourn, A.E. Metabolic diversification - Independent assembly of operon-like gene clusters in different plants. Science 2008, 320, 543-547. 31. Osbourn, A. Gene Clusters for secondary metabolic pathways: An emerging theme in plant biology. Plant Physiol. 2010, 154, 531-535.

503

23

ACS Paragon Plus Environment

Environmental Science & Technology

504

Figure Captions

505 506

Figure 1: Transmission electron micrograph of 20-nm silver nanoparticle (AgNP) suspensions

507

prepared in 0.5-strength Murashige and Skoog (MS) medium with polyvinylpyrrolidone

508

(molecular weight 40,000 Dalton, PVP40).

509 510

Figure 2: Relative biomass of A. thaliana plants exposed for 10 days to various concentrations

511

of silver nanoparticles (AgNPs) and silver ions (Ag+). Controls are non-exposed plants growing

512

in nutrient medium (MS) only and in nutrient medium containing PVP40 (PVP). Error bars

513

represent standard deviations between 20 biological replicates. Treatments resulting in

514

significantly different biomass (based on one-way ANOVA (p < 0.05) followed by Tukey's

515

multiple comparison tests) are shown by different letters.

516 517

Figure 3: Silver content measured in tissues of A. thaliana exposed to various concentrations of

518

silver nanoparticles (AgNPs) and silver ions (Ag+). Panel A: Leaves. Panel B: Roots. Controls

519

are non-exposed plants growing in nutrient medium (MS) only and in nutrient medium

520

containing PVP40 (PVP). Error bars represent standard deviations between 3 sets of pooled

521

biological replicates. Treatments resulting in significantly different biomass (based on one-way

522

ANOVA (p < 0.05) followed by Tukey's multiple comparison tests) are shown by different

523

letters.

524 525

Figure 4: Evaluation of microarray expression levels using RT-qPCR. Panel A: Log2 microarray

526

relative expression levels versus log2 RT-qPCR relative expression levels for exposure to silver

24

ACS Paragon Plus Environment

Page 24 of 31

Page 25 of 31

Environmental Science & Technology

527

nanoparticles (AgNPs). Panel B: Log2 microarray relative expression levels versus log2 RT-

528

qPCR relative expression levels for exposure to silver ions (Ag+). Error bars represent the

529

standard deviations between three biological replicates.

530 531

Figure 5: Major gene ontology (GO) process categories of genes up- (fold change > 2.0) and

532

down-regulated (fold change < 0.5) in A. thaliana plants exposed for 10 days to silver

533

nanoparticles (AgNPs) and silver ions (Ag+). Distribution of genes into GO categories was

534

performed using BLAST2GO® (GO level 2). Only categories containing at least 5% of the total

535

number of genes significantly expressed in response to the treatments are shown. Panel A:

536

Genes differentially expressed by exposure to silver nanoparticles (AgNPs). Panel B: Genes

537

differentially expressed by exposure to Ag+.

538 539

Figure 6: Major gene ontology (GO) functional categories of genes up- (fold change > 2.0) and

540

down-regulated (fold change < 0.5) in A. thaliana plants exposed for 10 days to silver

541

nanoparticles (AgNPs) and silver ions (Ag+). Distribution of genes in GO categories was

542

performed using BLAST2GO® (GO level 2). Only categories containing at least 5% of the total

543

number of genes significantly expressed in response to the treatments are shown. Panel A:

544

Genes differentially expressed by exposure to silver nanoparticles (AgNPs). Panel B: Genes

545

differentially expressed by exposure to Ag+.

546

25

ACS Paragon Plus Environment

Environmental Science & Technology

547

Figure 1

548

549 550 551

26

ACS Paragon Plus Environment

Page 26 of 31

on t C rol on M S t A gN rol P P 1. VP A gN 0 P mg / 2 A gN .5 L m P g A 5.0 /L gN m P g/ L 1 A gN 0 m P g 20 /L A g+ m 1 g/L A .0 m g+ 2 g/L A .5 m g+ 5. g/L A 0m g+ g /L 1 A 0m g+ g 20 /L m g/ L

C

Relative Biomass (%)

Page 27 of 31

552

Environmental Science & Technology

Figure 2

553

554

555

556

27

ACS Paragon Plus Environment

557

25

A. Leaf tissues

10 a

5 a a a a a

a

C on t C rol o M A ntro S gN lP P VP 1 A gN .0 P mg 2 / A gN .5 L P mg A 5.0 /L gN m g P 10 /L A gN m P g/ L 2 A 0m g+ 1. g/L A 0m g+ 2. g/L A 5m g+ 5. g/L 0 A g + mg 10 /L A g + mg 20 /L m g/ L

C on t C rol on M tr A S gN ol P P V 1 P A gN .0 P mg 2 / A gN .5 L P mg A 5.0 /L gN m P g/ L 1 A gN 0 m P g/ L 2 A 0m g+ g 1. /L A 0m g+ g /L 2. A 5m g+ g / 5. 0 L A g + mg 10 /L A g + mg 20 /L m g/ L

Environmental Science & Technology

b

200

20

a a

50

a a

0 a a

Treatment

559

560

561

28

ACS Paragon Plus Environment

Page 28 of 31

Figure 3

558

B. Root tissues

c b

f

150

15 100 e d

d c

b a

Treatment

e

b a b a d b

0 a

Page 29 of 31

562

Environmental Science & Technology

Figure 4

563

B. AG

A. SNP

6

Log2(Signal) - RT-qPCR

Log2(Signal) - RT-qPCR

6 4 2 0 -2 -4

4 2 0 -2 -4 -6

-6 -5

-4

-3

-2

-1

0

1

2

3

-5

4

-4

-3

-2

-1

0

1

2

Log2(Signal) - Microarray

Log2(Signal) - Microarray

564

565

566

29

ACS Paragon Plus Environment

3

4

Environmental Science & Technology

567

Figure 5

568

A

B

569 570

30

ACS Paragon Plus Environment

Page 30 of 31

Page 31 of 31

571

Environmental Science & Technology

Figure 6

572

573

574

31

ACS Paragon Plus Environment